DNA Repair and Free Radicals, New Insights into the Mechanism of Spore Photoproduct Lyase Revealed by Single Amino Acid Substitution
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m806503200
ISSN1083-351X
AutoresAlexia Chandor-Proust, Olivier Berteau, Thierry Douki, Didier Gasparutto, Sandrine Ollagnier de Choudens, Marc Fontecave, Mohamed Atta,
Tópico(s)Ammonia Synthesis and Nitrogen Reduction
ResumoThe major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(α-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed. The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(α-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed. Because of their chemical reactivity with regard to biological macromolecules (proteins, lipids, DNA, and others), free radicals are responsible for major damages when produced in large amounts within living cells (1Halliwell B. Gutteridge J. Free Radicals in Biology and Medicine. Oxford University Press, New York, NY2007Google Scholar). On the other hand, there has been an explosive growth in the number of enzymatic reactions found to proceed by mechanisms involving free radicals as intermediates (2Frey P.A. Hegeman A.D. Reed G.H. Chem. Rev. 2006; 106: 3302-3316Crossref PubMed Scopus (111) Google Scholar). This chemistry implies that enzymes have evolved delicate mechanisms to generate reaction-initiating primary radicals from stable substrates and to control these highly reactive species within the active sites so that they can undergo subsequent steps in the enzymatic process and do not get inactivated through radical coupling or harmful reactions with the protein itself or with oxygen, for example. This represents a fascinating issue in enzymology, and many mysteries of this chemistry remain to be unraveled. The spore photoproduct lyase enzyme (SPL) 4The abbreviations used are: SPLspore photoproduct lyaseSPspore photoproduce, 5-(α-thyminyl)-5,6-dihydrothymineAdoMetS-adenosylmethionine or S-adenosyl-l-methionine (SAM)AdoH5′-deoxyadenosylDTTdithiothreitolHPLChigh-performance liquid chromatographyMS/MStandem mass spectrometrywtwild typeRtretention timeTpTdinucleoside monophosphateTSΦpT5-(thiophenylmethyl)-2′-deoxyuridylyl-(3′,5′)thymidineTpTSΦthymidylyl-(3′,5′)-5-(thiophenylmethyl)-2′-deoxyuridine studied here is a nice illustration of how a radical chemistry is used for the cleavage of unreactive C–H and C–C bonds to repair DNA and how the control of intermediate radicals can be drastically lost by a single amino acid substitution. During exposure to UV light of bacterial spores, a dormant form produced by some bacteria such as Bacillus and Clostridium species, dimerization of adjacent thymine bases in DNA results in the specific formation of 5-(α-thyminyl)-5,6-dihydrothymine, the so-called spore photoproduct SP (Scheme 1A), a lesion that can block replication and transcription and is thus potentially lethal, mutagenic, and cytotoxic (3Donnellan Jr., J.E. Setlow R.B. Science. 1965; 149: 308-310Crossref PubMed Scopus (147) Google Scholar,4Varghese A.J. Biochem. Biophys. Res. Commun. 1970; 38: 484-490Crossref PubMed Scopus (120) Google Scholar). Sporulating bacteria are extremely resistant to UV radiation, because they use SPL early in the spore germination cycle, to very efficiently and specifically revert SP to two unmodified thymines (Scheme 1A) (5Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (334) Google Scholar, 6Slieman T.A. Rebeil R. Nicholson W.L. J. Bacteriol. 2000; 182: 6412-6417Crossref PubMed Scopus (47) Google Scholar, 7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). spore photoproduct lyase spore photoproduce, 5-(α-thyminyl)-5,6-dihydrothymine S-adenosylmethionine or S-adenosyl-l-methionine (SAM) 5′-deoxyadenosyl dithiothreitol high-performance liquid chromatography tandem mass spectrometry wild type retention time dinucleoside monophosphate 5-(thiophenylmethyl)-2′-deoxyuridylyl-(3′,5′)thymidine thymidylyl-(3′,5′)-5-(thiophenylmethyl)-2′-deoxyuridine The SPL enzymatic mechanism has been addressed only recently, when pure preparations of SPL became available, and the following data are consistent with a radical mechanism initially proposed by T. P. Begley, on the basis of model compounds (8Mehl R.A. Begley T.P. Organic Lett. 1999; 1: 1065-1066Crossref PubMed Scopus (53) Google Scholar). First, SPL is a "Radical-SAM (S-adenosyl-methionine)" iron-sulfur enzyme (9Sofia H.J. Chen G. Hetzler B.G. Reyes-Spindola J.F. Miller N.E. Nucleic Acids Res. 2001; 29: 1097-1106Crossref PubMed Scopus (805) Google Scholar). This class of enzymes uses a 5′-deoxyadenosyl radical, produced from reductive cleavage of AdoMet, to initiate the reaction (Scheme 1B) (10Fontecave M. Mulliez E. Ollagnier-de-Choudens S. Curr. Opin. Chem. Biol. 2001; 5: 506-511Crossref PubMed Scopus (83) Google Scholar, 11Frey P.A. Hegeman A.D. Ruzicka F.J. Crit. Rev. Biochem. Mol. Biol. 2008; 43: 63-88Crossref PubMed Scopus (441) Google Scholar). Second, experimental evidence of label transfer to AdoMet from SP in DNA specifically 3H-labeled at C-6 indirectly shows that repair is initiated by abstraction of the C-6 hydrogen atom of SP by the 5′-deoxyadenosyl radical (12Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (105) Google Scholar) (Scheme 1B). The subsequent steps have been less characterized, but it is likely that C–C bond homolytic cleavage follows to give an allyl-type radical together with a first repaired thymine. The last step of the reaction has been proposed to involve hydrogen atom transfer from 5′-deoxyadenosine (AdoH) to the thymine monomer radical (12Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (105) Google Scholar). Preliminary experiments have indeed shown some label transfer from [5′-3H]AdoMet into thymine monomers, using a DNA substrate containing the SP lesion, thus supporting the mechanism shown in Scheme 1B (12Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (105) Google Scholar, 13Buis J.M. Cheek J. Kalliri E. Broderick J.B. J. Biol. Chem. 2006; 281: 25994-26003Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this report, we provide evidence for the involvement of the allyl-type radical during in vitro B. subtilis SPL-dependent repair reaction. We took advantage of using pure preparations of a dinucleoside monophosphate substrate named SPTpT in the following (Scheme 2), in one hand, and, in another hand, an SPL mutant in which Cys-141, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has been changed into alanine (14Fajardo-Cavazos P. Rebeil R. Nicholson W.L. Curr. Microbiol. 2005; 51: 331-335Crossref PubMed Scopus (30) Google Scholar). We were indeed intrigued by this observation, because Cys-141 is not a ligand of the [Fe-S] cluster present in the active site, in contrast to cysteines 91, 95, and 98 of the CysXXXCysXXCys motif characteristic of Radical-SAM enzymes. Implications of the data reported here regarding the reaction mechanism and the role of Cys-141 are discussed. Materials—Strains: Escherichia coli DH5α was used for routine DNA manipulations. E. coli Tuner (DE3) was used for enzymes overexpression. Enzymes, oligonucleotides, and culture media were purchased from Euromedex (Strasbourg, France). Plasmid DNA purification kit, Qiaprep™, was from Qiagen. DNA sequencing was performed by Genome Express (Grenoble, France). Cloning and Construction of Mutant C141A SPL-overexpressing Plasmid—The plasmid containing the SplB gene, encoding wild-type N-terminal 6His-tagged SPL (pT7-SPL6H), was obtained as previously described (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). A site-directed mutagenesis was performed to change cysteine 141 into an alanine using QuikChange site-directed mutagenesis kits from Stratagene according to the manufacturer's protocol. The mutant plasmid was entirely sequenced to ensure that no error was introduced during PCR reaction. The plasmid was then named pT7-SPLC141A. Protein Expression—For both wt SPL and SPL-C141A, expression was conducted in LB medium in the E. coli Tuner (DE3), and proteins were purified under aerobic conditions as previously described (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Iron and Sulfide Binding to WT and C141A B. subtilis SPL—[Fe-S] cluster reconstitutions of wt and C141A B. subtilis SPL were carried out under strictly anaerobic conditions in a glove box containing <2 ppm O2 as previously described (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Production of SPTpT Substrates—The spore photoproduct of the dinucleoside monophosphate thymidylyl-(3′,5′)thymidine was prepared as previously described (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). SP-Lyase Activity—To assay SPL activity we followed the same procedure as for Wild-type SPL (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), using 2.5 mm DTT, 1.5 mm dithionite, and 1.5 mm AdoMet. At each time point (0, 15 30, 60, 120, 215, and 280 min) 20 μl and 10 μl of the solution were transferred into Eppendorf tubes, and the reaction was stopped by flash-freezing in liquid nitrogen. Conversion of the spore photoproduct (SPTpT) into the unmodified dinucleoside monophosphate (TpT) and other products in SPL-treated samples was quantified by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS), using the same conditions as for samples treated by wt SPL (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). HPLC-MS Analysis—HPLC-MS/MS analyses were carried out on an API 3000 tandem mass spectrometer associated to a Series 1100 Agilent chromatography system. An Uptisphere ODB reversed-phase octadecylsilyl silica gel column (3-μm particle size, 150 × 2 mm inner diameter, Interchim, Montluçon, France) was used in all analyses. A gradient of acetonitrile in 2 mm aqueous triethylammonium acetate was used for most analyses, which were carried out in the negative electrospray ionization mode. These conditions were applied for TpT (retention time (Rt) 26.6 min), SPTpT (Rt 19.9 min), the SO2 derivatives of TpT (TpTSO2 Rt 21.7 min and TSO2pT Rt 22.3 min), the hydroxylated derivatives of TpT (TpTOH Rt 25.6 min and TOHpT Rt 24.3 min). Experiments were also carried out in the positive electrospray ionization mode. For that purpose, acetate ammonium was used as the HPLC buffer. Under these conditions, the retention times were the following: TpTSO2 18.3 min, TSO2pT 19.2 min, 5′-deoxyadenosyl (AdoH) 5.1 min, and AdoMet 3.1 min. The chromatograms were recorded either in the MS1 (full spectra without fragmentation), SIM (selected ion monitoring without fragmentation), MS2 (full spectra of fragment arising from a specific parent ion), and MRM (multiple reaction monitoring, specific for transition corresponding to compounds of interest) modes. SAM Reductase Activity—The cleavage of AdoMet was monitored by HPLC from the formation of AdoH as previously described (15Pierrel F. Douki T. Fontecave M. Atta M. J. Biol. Chem. 2004; 279: 47555-47563Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Protein Analysis—Protein concentration (by monomer) was determined by the method of Bradford (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217529) Google Scholar). Protein-bound iron (17Fish W.W. Methods Enzymol. 1988; 158: 357-364Crossref PubMed Scopus (535) Google Scholar) and labile sulfide (18Beinert H. Anal. Biochem. 1983; 131: 373-378Crossref PubMed Scopus (403) Google Scholar) concentrations were determined according to standard procedures. Preparation and Light-induced Decomposition of Thiophenyl Derivatives of TpT—5-(Thiophenylmethyl)-2′-deoxyuridylyl-(3′,5′)thymidine (TSΦpT) and thymidylyl-(3′,5′)-5-(thiophenylmethyl)-2′-deoxyuridine (TpTSΦ) were prepared as previously described (19Romieu A. Bellon S. Gasparutto D. Cadet J. Organic Lett. 2000; 2: 1085-1088Crossref PubMed Scopus (94) Google Scholar). In a first series of experiments, solutions containing 40 μm TpTSΦ or TSΦpT together with 1.5 mm Na2S2O4 were kept overnight in an anaerobic glove box. Samples were then exposed for 5 min to UV-C radiation (254 nm) emitted by a germicidal lamp. A second series of irradiations was performed with aerated solutions of either TpTSΦ or TSΦpT. Samples were then analyzed by HPLC-MS/MS without further treatment. Elution was monitored in the MS2 mode. Parent ions were set at m/z 609 and 561 in the negative mode and 611 in the positive mode. UV-visible Absorption Spectroscopy—UV-visible absorption spectra were recorded with a Cary 1 Bio (Varian) spectrophotometer. EPR—Reconstituted SPL-C141A (187 μm, 4.2 iron/protein) were reduced with 2 mm dithionite under anaerobic conditions for 20 min and frozen inside the glove box. X-Band EPR spectra were recorded on a Brucker Instruments ESP 300D spectrometer equipped with an Oxford Instruments ESR 900 flow cryostat (4.2–300 K). Spectra were quantified under non-saturating conditions by double integration against a 1 mm Cu-EDTA standard. Mössbauer Spectroscopy—For Mössbauer measurements, reconstituted SPL-C141A (9.6 mg and 4.2 iron/protein) was prepared as described above in a final volume of 400 μl. The protein solution was transferred into a Mössbauer cup and frozen in liquid nitrogen. 57Fe-Mössbauer spectra were recorded at zero magnetic field on a spectrometer operating in constant acceleration mode using an Oxford cryostat that allowed temperatures from 1.5 to 300 K and a 57Co source in rhodium. Isomer shifts are reported relative to metallic iron at room temperature. Cloning, Overexpression, and Purification of SPL C141A Variant—The pT7-SPL-C141A plasmid, obtained as described under "Experimental Procedures," was used to transform E. coli Tuner (DE3) strain for production of a protein with a His tag at the N-terminal end and an alanine at position 141 in place of a cysteine. A His tag at this extremity is not detrimental to wild-type (wt) SPL enzyme activity (20Rebeil R. Sun Y. Chooback L. Pedraza-Reyes M. Kinsland C. Begley T.P. Nicholson W.L. J. Bacteriol. 1998; 180: 4879-4885Crossref PubMed Google Scholar). The SPL-C141A mutant protein could be obtained in pure form, with traces of protein-bound iron and acid-labile sulfide. Reconstitution of the [Fe-S] cluster yielded a SPL-C141A protein containing ∼3–3.5 iron and 3.5–4 sulfur atoms per monomer, in agreement with the presence of a [4Fe-4S] cluster. The UV-visible spectrum, with two broad bands at 330 and 420 nm, of the reconstituted mutant as well as the EPR spectrum of the dithionite-reduced protein, with a signal characteristic for a S = 1/2 [4Fe-4S]+1 species, were similar to those of wt SPL (supplemental Figs. S1 and S2). The [Fe-S] cluster was further investigated by Mössbauer spectroscopy using a protein sample (277 μm, 4.01 iron/monomer) reconstituted with 57Fe and sulfide as previously described in the case of wt SPL (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The spectra recorded at 78 K and 4.2 K were dominated by a quadrupole doublet, the parameters of which (δ ∼ 0.44(1) mm.s-1, ΔEQ = 1.07(2) mm.s-1) clearly corresponded to a [4Fe-4S]2+ cluster, accounting for ∼73% of total Fe (supplemental Fig. S3). Extra iron was in the form of high spin ferrous iron, as observed in the case of wt SPL. In Vitro SPTpT Repair Activity of the Wild-type SPL Enzyme—We assayed SPL activity using the SPTpT dimer, shown in Scheme 2, as a pure substrate. SPTpT is produced by UV irradiation of the unmodified dinucleoside monophosphate TpT. The methylene bridge in SP corresponds to the methyl group of the 3′-end thymine (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This synthetic compound is identical to that released from DNA extracted from UV-irradiated spores as shown by their identical HPLC retention time and fragmentation mass spectrometry features (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Use of this substrate facilitated monitoring of the repair reaction, because assay samples could be directly submitted to HPLC-MS/MS analysis at the end of the incubation time, for separation, identification, and quantitation of SPTpT and TpT (Scheme 2). We recently reported that this substrate is totally transformed into TpT under standard assay conditions, using wt SPL (7Chandor A. Berteau O. Douki T. Gasparutto D. Sanakis Y. Ollagnier-de-Choudens S. Atta M. Fontecave M. J. Biol. Chem. 2006; 281: 26922-26931Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). For sake of comparison with the SPL-C141A enzyme, a standard highly reproducible wt SPL time-dependent repair experiment is shown in Fig. 1A. The assay mixture contained a pure preparation of reconstituted wt SPL (1 μm) together with 1.5 mm dithionite as a reducing agent, 1.5 mm AdoMet and 10 μm SPTpT in DTT-containing buffer at pH 8 under strict anaerobic conditions. The repair reaction was then assayed at time intervals by HPLC-MS/MS for its content in SPTpT and TpT. Fig. 1A shows that a quantitative conversion of SPTpT to TpT was achieved in 90% yield) of the reaction is a modified dinucleoside monophosphate containing one thymine and one thymine with a sulfinate moiety compound B (Scheme 4). We also demonstrate that the sulfinate group: (i) is attached to the methyl carbon of the base; (ii) is exclusively present in the thymine of the 3′-end of the dinucleoside monophosphate; and (iii) is derived from dithionite, the reducing agent present in the in vitro assay mixture. These results are interpreted by the mechanism shown in Scheme 4 for the repair of SPTpT catalyzed by the wt SPL and the mutant SPL-C141A enzyme. Because both enzymes are able to generate products lacking the initial covalent bond between the bases it is likely that the first steps of the reaction, during which the cross-link is broken, are the same and that the substitution of cysteine 141 into alanine is not detrimental to substrate binding in the active site of the protein and reacting with the cofactor. This is consistent with the fact that the SPL-C141A enzyme chelates a [4Fe-4S] cluster with high yield, as shown by Mössbauer spectroscopy, and catalyzes the reductive cleavage of AdoMet in the absence of substrate, as does wt SPL (our results and Refs. 21Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (83) Google Scholar, 22Pieck J.C. Hennecke U. Pierik A.J. Friedel M.G. Carell T. J. Biol. Chem. 2006; 281: 36317-36326Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Thus, in both cases the reaction starts with the reductive cleavage of AdoMet by the reduced cluster generating the 5′-deoxyadenosyl radical, which then abstracts one of the H atoms at the methylene C-6 position. This primary radical undergoes a β-scission leading to the homolytic cleavage of the C–C bond linking the two thymines generating an allylic radical. Differences between the two enzymes appear in the last step of the reaction (Scheme 4). Our data suggest that the substitution of Cys-141 into alanine is responsible for a deficient control/protection of the allylic radical intermediate, which then is efficiently trapped by the SO2·¯ radicals generated in solution by dithionite, through a radical-radical coupling reaction producing the sulfinate compound (Scheme 4). This mechanism implies two key radical intermediates. The involvement of the first one, derived from H atom abstraction at C-6 methylene by the 5′-deoxyadenosyl radical Ado·, has been demonstrated by DNA labeling experiments, using the wt enzyme, previously reported (12Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (105) Google Scholar). The involvement of the second one, the allylic radical precursor of the repaired product, is now indirectly proven by the experiments reported here using the SPL-C141A mutant enzyme. The latter in that respect proved remarkable as it indeed unexpectedly allowed an efficient trapping of this second radical by dithionite, which could be easily monitored by HPLC/mass spectrometry. At this stage it is not known how Cys-141 provides the radical control/protection mechanism, but two possibilities, requiring further investigation, are briefly discussed in the following. The first hypothesis implies Cys-141 as an H atom donor to the intermediate allylic radical. Such a reaction is thermodynamically reasonable, considering that the energy of the S–H bond of a cysteine residue is slightly larger than to that of the C–H bond of an allyl group (25Stubbe J. van Der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1368) Google Scholar). Substitution of Cys-141 into alanine thus would result in a further stabilization of the intermediate allylic radical, thus facilitating its coupling with highly reactive free radicals in the environment such as those produced by dithionite in the in vitro experiments. To be catalytic, the resulting protein-bound cysteinyl radical generated in SPL after TpT formation needs to be converted back to a normal cysteine. This reaction implies an H atom donor, which has not been identified but might be DTT in vitro. A second hypothesis implies that cysteine 141 participates in the stabilization of a specific structure of the active site, which prevents the intermediate radical from reacting with exogenous free radicals. In this model, the wt enzyme has its active site in a "closed" conformation because of the presence of Cys-141 and the allylic radical does not react with the dithionite-derived radicals present in the in vitro reaction mixture. Instead, it reacts almost exclusively with an H atom donor, by H atom radical abstraction, to generate the final repaired TpT product. In the case of the SPTpT substrate used here, because AdoH is excluded, the nature of the H atom donor is unknown. Considering that with DNA substrate AdoH is the direct H atom donor (Scheme 1B), we consider the second hypothesis as more likely. Obviously a structural characterization of SPL is required to substantiate at the molecular level such a hypothesis. In conclusion, our data confirm that a single substitution of cysteine-141 into alanine has a major impact on the SPL-dependent repair reaction and are consistent with the high sensitivity of bacterial spores containing a C141A SPL mutant enzyme to irradiation, as previously reported (14Fajardo-Cavazos P. Rebeil R. Nicholson W.L. Curr. Microbiol. 2005; 51: 331-335Crossref PubMed Scopus (30) Google Scholar). How this amino acid residue contributes to tightly control the outcome of highly energetic and reactive radical intermediates during catalysis remains to be understood. Furthermore, the mutant enzyme studied here has allowed trapping of one of the key catalytically competent free radical intermediates, providing new insights into the mechanism of SP repair by the SPL enzyme. We thank Y. Sanakis (Institut of Materials Science, National Center for Scientific Research, Attiki, Greece) for Mössbauer analysis on the SPL-C141A enzyme and P. Frey (Enzyme Institute, University of Madison, Madison, WI) for providing [5′-(2H)2]SAM. Download .pdf (.12 MB) Help with pdf files
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